TECHNICAL FIELD
[0001] This invention is related to arabinonucleic acid and the use of this novel polynucleotide
probe in DNA or RNA hybridization assays and especially to probes containing a binding
site in every constituent nucleotide. This permits the attachment of a detection moiety
to each nucleotide of the probe and thus leads to improved detection sensitivity.
BACKGROUND ART
[0002] Hybridization probes used in DNA and RNA assays are labeled in some fashion to facilitate
detection of the duplex after the probe has hybridized with a complementary strand
of -target- DNA or RNA in the sample under analysis. Most commonly, the probe can
be labeled by enzymatically incorporating radiolabeled nucleotides on the 3' or 5'
terminus of the probe. [A.M. Maxam et al.. Meth. Enzymol. Volume 65, 499-560 (1980)].
Alternatively, higher levels of radiolabeled nucleotides can be incorporated by nick
translation [P.W.J. Rigby et al.. J. Mol. Biol. Volume 1
13, 23
7-251 (1977)]. This latter method possesses the inherent advantage of being able to
incorporate one radioactive phosphorus per constituent nucleotide in the probe and
thus assays using these probes are characterized by the most sensitive detection limits
in hybridization assays. The obvious disadvantages of these probes are the hazards
and inconveniences associated with the use of radioisotopes.
[0003] A second type of label involves enzymatically synthesizing a probe with a mixture
of nucleotides containing a biotinylated pyrimidine base (uracil or adenosine) incorporated
into one of the nucleotides [e.g., P. R. Langer et al., Proc. Natl. Acad. Sci. USA
Volume 78. 6633-6637 (1981)]. Alternatively, biotinylated histone H1 proteins can
be chemically crosslinked with bases in DNA to yield a labeled probe [M. Renz, EMBO
J. Volume 2(6), 817-822 (1983)]. After hybridization of the probe to a complementary
strand of target DNA, the hybrid is treated with a reporter molecule attached to avidin
which binds tenaciously to the biotin moieties in the probe. The reporter can be either
an opaque polymeric microsphere bound to avidin [O. C. Richards et al.. Proc. Natl.
Acad. Sci. USA Volume 76(2). 676-680 (1979)] or avidin-ferritin [A. Sodja et al..
Nucl. Acids Res. Volume 5(2), 385-
401 (1978)]. Each of these can be visualized via electron microscopy. Alternatively,
the reporter may be an avidin-enzyme complex which when treated with the enzyme's
substrate, will yield a visually detectable colored product [J. J. Leary et al.. Proc.
Natl. Acad. Sci. USA. Volume 80 4045-4049 (1983)]. Assays based on these probes obviate
the use of radioactive materials: however, only about 2% of the nucleotides in a given
probe molecule can be derivatized with a biotin moiety without reducing the specificity
of the probe for its complementary strand of sample DNA. This reduction in specificity
arises from modification of the ability of complementary bases to pair properly because
of the presence of the biotin moiety. The reduced incorporation of label in these
probes relative to radioactive probes results in a poorer detection sensitivity.
[0004] A third type of label is an enzyme directly attached to the probe such that after
hybridization the enzyme in the hybrid is treated with substrate to yield a colored
product. The enzyme can be bonded directly to bases in the probe [M. Renz et al..
Nucl. Acids Res., Volume 12(8). 3435-3444 (1984)] or to bases in another strand of
DNA complementary to a length of oligonucleotide chemically attached to the end of
the probe [J. G. Woodhead et al., Biochem. Soc., Trans., Volume 12(2). 279-280 (1984)].
In the latter case. the probe is hybridized to complementary target DNA and this hybrid
is treated with the oligonucleotide containing the enzyme labels. This strand of labeled
oligonucleotide binds with the single-stranded tail of complementary DNA which had
been chemically attached to the end of the probe. Visualization of the enzyme reporters
is accomplished by conversion of the enzyme
's substrate to colored product. The primary difficulties associated with this procedure
arise from the low level of enzyme attachment and the loss of enzyme activity when
subjected to the stringent conditions (e.g. elevated temperature, nonaqueous solvent)
typically used in the hybridization protocol. A secondary difficulty associated with
the latter of these labeling methods is the possibility that the synthetic strand
of oligonucleotide containing the enzyme label might be complementary to a sequence
of naturally-occurring nucleotides in the sample. Thus, in addition to pairing with
the sequence of bases attached to the end of the hybridization probe, the enzyme-labeled
oligonucleotide may bind nonspecifically to natural sequences of complementary bases
and thus lead to background problems in samples used as negative controls or blanks.
[0005] A fourth type of label involves coupling a fluorophore either to bases in the constituent
nucleotides of the probe [C. H. Yang et al.. J. Biochem. Volume 13. 3615-3620 (1974)]
or to the 3'-terminus
[R. W. Richardson et al.. Nucl. Acids Res. Volume 11(8). 6167-6184 (1983)] or to the
5' terminus [C. H. Yang et al.. Arch. Biochem. Biophys., Volume 155. 70-81 (1973)]
of the probe. As above, the sensitivity of these methods is limited by the small number
of labels which can be incorporated into each copy of the probe.
[0006] A fifth type of label involves the use of an antibody directed against some antigenic
determinant in the probe or in the target-probe duplex. In the former case, an antigenic
determinant (e.g., biotin, bromine. N-acetoxy-N-2-acetylaminofluorene) is covalently
coupled to bases in the nucleotide of the probe [e.g.. L. Manuelidis et al.. J. Cell
Biol.. Volume 95, 617-625 (1982)]. These antigenic determinants may interfere with
specific hybridization and limit the utility of the probe. In the latter case. the
double-stranded DNA-RNA hybrid itself is immunologically distinguishable from DNA-DNA
and RNA-RNA duplexes [G. T. Rudkin et al., Nature. Volume 265. 472-473 (1977)]. The
antigenic determinant in the probe-target duplex was detected with an antibody- fluorophore
conjugate and fluorescence microscopy. The labeled probe can also be detected with
antibodies to which an enzyme (L. Manuelidis et al., op. cit.) or colloidal gold [N.
J. Hutchinson et al.. J. Cell Biol.. Volume 95. 609-618 (1982)] has been conjugated.
Again, the ultimate sensitivity is limited by the number of labels available to detect
a hybridization event.
[0007] The instant invention overcomes the limitation of the prior art by incorporating
the maximal number of nonradioactive labels, one per nucleotide. into each probe with
negligible effect on hybridization efficiency or specificity.
DISCLOSURE OF THE INVENTION
[0008] This invention involves a new nucleic acid, arabinonucleic acid.
[0009] The method of this invention for identifying nucleic acid sequences comprises the
steps of:
(a) rendering the target nucleic acids single-stranded;
(b) immobilizing the single-stranded nucleic acids onto a support:
(c) allowing said single-stranded nucleic acids to hybridize with a single-stranded
arabinonucleic acid probe;
(d) washing said support to remove arabinonucleic acid not incorporated into the hybrid
formed on the support: and
(e) determining the presence of arabinonucleic acid in the hybrid formed on the support
by contacting it with an anti-arabinose antibody-label conjugate and detecting said
label.
DESCRIPTION OF THE INVENTION
[0010] This invention involves the synthesis and use of a novel hybridization probe for
DNA and RNA assays. This new probe, arabinonucleic acid (ANA), has an arabinose sugar
replacing the conventional ribose or deoxyribose sugars found in RNA and DNA, respectively.
This uncommon sugar provides binding sites for an anti-arabinose antibody-label which
can be used to detect selectively the arabinose sugar and thus any probe containing
the sugar. The sugar arabinose is found only in this synthetic probe and not in any
naturally-occurring DNA or RNA and, therefore, the detection antibody will bind only
to the arabinose moieties in the probe.
[0011] The probe can be synthesized either chemically or enzymatically. Chemical synthesis
involves the introduction of protective groups on the 2'. 3' and 5' carbon atoms of
the arabinose sugar in a nucleoside containing arabinose instead of ribose or deoxyribose.
Such nucleosides are commercially available. The chemical nature and method of attaching
the protective groups has been developed for ribonucleo- sides and deoxyribonucleosides
[G. H. Hakimeliah et al.. Can. J. Chem., Volume 60. 1106-1113 (1982)] and can be adapted
for protection of the corresponding positions in arabinonucleosides [K. K. Ogilvie
et al., Can. J. Chem.. Volume 61, 1204-1212 (1983)]. Once the protection of the arabinonucleosides
has been achieved. the chemical synthesis of the arabinonucleic acid probe can then
proceed in the manner used for DNA and RN
A probes, in that the correct nucleotides will be sequentially linked to form a chain
of nucleotides which is complementary to a sequence of nucleotides in target DNA or
RNA in the sample under analysis (M. H. Caruthers et al., in Genetic Engineering,
J. Setlow. ed.. Vol. 4, 1-17. 1982).
[0012] Nucleic acids to be analyzed have many sources. These include clinical specimens,
various microorganisms such as bacteria, viruses, chlamydia, rickettsia. mycoplasma
and protozoa, plants, among others. Extraction is one common method for collecting
the nucleic acids from their sources for hybridization assays. The protocol for use
of the ANA probes of this invention is much like that in conventional hybridization
procedures. The target DNA or RNA is first rendered single-stranded and then immobilized
onto a support. The immobilized single-stranded nucleic acids are then treated with
the arabinonucleic acid probe complementary to a sequence of bases in the target.
Hybridization of the arabinonucleic acid probe with the complementary sequence of
bases is allowed to occur. After washing to remove excess unhybridized ANA probe,
the probe-target hybrid is treated with an anti-arabinose antibody-label conjugate.
[0013] The antibody itself is raised against the C2 chiral site in arabinose or the C2 site
in conjunction with other antigenic determinants in the arabinose or ANA structures
[Okabayashi et al., Cancer Research Volume 37, 619-624 (1977)]. The antibody-conjugate
can be prepared by conventional means. The label can be enzymatic, fluorescent, chemiluminescent
or macromolecular, the preferred one being enzymatic.
[0014] This antibody-label conjugate specifically binds to the arabinose sugar but not to
ribose or deoxyribose present in native RNA or DNA. After treatment such as washing
to remove excess, unbound antibody-enzyme conjugate from the system it is treated
with the enzyme's substrate to produce a detectable signal such as a colored product,
indicating the presence of the target nucleic acid in the sample. Labels other than
enzymes can. of course, be detected appropriately.
[0015] Alternatively, one can envision other binding reagents, such as a lectin. which could
be conjugated to an enzyme or other labels. The assay protocol for such a conjugate
would be similar to that above.
[0016] Arabinonucleic acid probes have several unique advantages. Since one arabinose binding
site can be incorporated into each nucleotide of the hybridization probe, each nucleotide
is capable of having an anti-arabinose antibody-label bound to it to achieve labeling
at the levels usually obtainable with radioisotopes. At the same time, the hazards
of radioactive materials are avoided. In addition, these fully labeled probes will
not undergo the degradation that radiolabeled probes experience due to lysis by the
high-energy decay products of the radiolabels. Furthermore, since detectability of
the ANA relies upon the inverted chirality about the C2 carbon atom in the sugar backbone
of the probe, and since the chemical bonds about the C2 carbon atom are not involved
in determining either the intra- or inter-strand structure of the probe or the probe-target
hybrid, specificity of the probe and the stability of the probe-target hybrid are
not expected to be affected significantly. This higher degree of labeling of the ANA
probe, achieved without altering the specificity of the probe for its complementary
sequence of bases, makes the probes of this invention superior to the chemically derivatized
probes of the prior art. Finally, the arabinose in the probe can be specifically detected
using an antibody-enzyme conjugate. The advantage of the use of an enzyme label derives
from the high degree of signal magnification due to the large turnover of substrate
to product. Thus, use of antibody-enzyme conjugates to detect arabinose possesses
not only the advantage of the high degree of probe labeling characteristic of radiolabeled
probes but also the signal magnification of an enzyme-based nonradiometric assay.
Example 1
A. Chemical Synthesis of Protected Arabinonucleotides for Probe Synthesis
[0017] Probe synthesis through chemical polymerization of nucleotides requires the availability
of at least four protected and activated arabinonucleotides containing the bases guanine,
cytosine, adenine, and uracil or thymine. Each of these protected nucleotides can
be prepared from the corresponding nucleosides, which are commercially available.
[0018] The addition of protective and activating groups to a nucleoside will be illustrated
for the preparation of the protected and activated nucleoside arabinouracil (araU).
The other arabinonucleosides were protected and activated in an identical manner:
in addition. nucleosides containing the bases cytosine and adenine required protection
on the base itself in the form of a benzoyl group while guanine required an isobutyryl
protecting group.
[0019] First, a dimethoxytrityl (DMT) group was introduced at the hydroxyl on the 5
1-carbon of arabinose. This was done by adding 7.75 mmoles of dimethoxytrityl chloride
(DMTCI) in eight equal portions at 1-hour intervals to 6.5 mmoles of B-D-arabinouracil
dissolved in 30 mL pyridine at -5°C. One hour after the final addition, the reaction
mixture was poured into ice water to destroy excess dimethoxytrityl chloride and reduced
to a gum at approximately 25°C on a rotary evaporator. The product. 5'-DMT araU, was
isolated from the reaction mixture on a column of Merck Kieselgel 60 silica gel eluted
with a 95/5 mixture of chloroform/methanol. Yields of 90-95% were obtained.
[0020] A tert-butyldimethysilyl group (TBDMS) was then introduced at the hydroxyl group
on the 3
1-carbon of arabinose in the arabinonucleoside. 5'-DMT araU, 5.5 mmoles, prepared as
above, was dissolved in dimethoxyethane (110 mL) and 44 mmoles of triethylamine was
added. Next, 16.5 mmoles of silver nitrate was added and the mixture stirred for 1
hour at room temperature. Then, 16.5 mmoles of tert-butyldimethylsilyl chloride was
added and this reaction mixture stirred for 5 hours at room temperature. The reaction
mixture was filtered into a 10% solution of sodium bicarbonate and the aqueous mixture
was extracted twice with methylene chloride. The organic extract was evaporated to
dryness and 5'-DMT.3'-TBDMS araU was isolated in 75% yield from a Merck Kieselgel
60 silica gel column eluted with ethyl acetate.
[0021] A benzoyl (Bz) protecting group was introduced at the hydroxyl on the 2'-carbon of
arabinose in the nucleoside by dissolving 5.4 mmoles of the previously prepared 5'-DMT.3'-TBDMS
araU in 40 mL of pyridine and cooling to -45°C. An excess (6.6 mmoles) of benzoyl
chloride in methylene chloride was added dropwise to the stirred reaction mixture
which was held at -45°C for 30 minutes after addition was complete. The reaction mixture
was warmed and held at -20°C for 2 hours. After addition of water to hydrolyze unreacted
benzoyl chloride, the mixture was reduced to a gum on a rotary evaporator and 5'-DMT.3'-TBDMS.2'-Bz
araU was isolated on a silica gel column eluted with a 50/50 mixture of toluene and
ethyl acetate.
[0022] The temporary blocking group (TBDMS) on the hydroxyl on the 3'-carbon was removed
by treating 0.78 mmole of 5'-DMT.3'-TBDMS.2'-Bz araU with 2.5 mmoles of 1M tetrabutylammonium
fluoride (in THF) at 25°C for 0.5 hour. The 5'-DMT.2'-Bz araU was isolated from a
silica gel column eluted with ethyl acetate.
[0023] The final step in the preparation of the nucleotide involved the activation of the
hydroxyl group on the 3'-carbon with a phosphine. 5'-DMT.2'-Bz araU (77 µmoles) was
dissolved in 0.23 mL of methylene chloride containing 48 uL of diisopropylethylamine.
Next. 31 µL of N,N-diisopropylmethylphosphonamidic chloride was added via syringe
to the stirred reaction mixture at 20°C. After 15 minutes, the reaction mixture was
diluted with ethyl acetate and extracted with aqueous sodium bicarbonate (saturated).
The organic layer was separated from the aqueous bicarbonate, dried with Na
2SO
4 and reduced on a rotary evaporator to yield the desired activated and protected nucleotide.
B. Probe Synthesis
[0024] It is envisioned that synthesis of the oligomeric ANA probe will follow the procedures
developed for production of DNA probes using similarly protected and activated deoxynucleotides
(M. H. Caruthers, et al., in Genetic Engineering. J. Setlow, ed. Volume 4, pp. 1-17).
According to these procedures, the first step in the synthesis would be the addition
of a starter derivatized arabinonucleoside to a silanol- derivatized silica support.
This will be done by reacting the appropriate 5'DMT.2'-Bz arabinonucleoside with succinic
anhydride. The succinylated arabinonucleoside would be converted to the p-nitrophenyl
ester by reaction with p-nitrophenol and dicyclohexyl carbodiimide. The activated
nucleoside will be reacted with aminopropyl-derivatized silica gel in a mixture of
dimethylformamide, dioxane, and triethylamine. Unreacted silanol groups on the silica
will be blocked by reaction with acetic anhydride. These steps would result in the
silica gel having the nucleoside attached to it at the 3'-end and. therefore, this
nucleoside will be at the 3'-end of the probe to be synthesized.
[0025] The next nucleotide can then be added to the nucleoside attached to the silica support.
The silica- nucleoside product prepared above will be treated with p-toluenesulfonic
acid in acetonitrile to remove the acid-labile dimethoxytrityl group from the hydroxyl
group on the 5'-carbon atom of the nucleoside attached to the silica support. The
nucleoside on the support will be condensed with the appropriate arabinonucleotide
phosphoramidite to add the next nucleotide to the 5'-end of the growing probe. This
reaction will be carried out in the presence of tetrazole in dry acetonitrile to facilitate
the condensation reaction. The unreacted 5'-hydroxyl group of the arabinonucleoside
on the support will be blocked by treatment, for 1-2 minutes, with acetic anhydride
in dimethylaminopyridine. The phosphite triester linkage formed by the preceding condensation
will be oxidized to a phosphate ester by treatment with a mixture of iodine and 2.6-
lutidine in aqueous tetrahydrofuran for 1-2 minutes. This step concludes the addition
of a nucleotide to the growing probe. Additional nucleotides can be added by repeating
the above reaction sequence starting with the removal of the protective group from
the 5'-hydroxyl in arabinose on the 5
1-end of the probe.
[0026] When the synthesis of the probe sequence is complete, the oligomeric probe will be
treated with triethylamine and thiophenol in dioxane to convert the phosphate triesters
in each nucleotide linkage to phosphate diesters. The probe then will be cleaved from
the silica support by treatment with concentrated ammonium hydroxide at 20
*C for 3 hours. This treatment will also remove the benzoyl protecting groups from
the 2
1-hydroxyl groups in each arabinose moiety in the probe and any protecting groups from
the bases. The oligomeric ANA probe will be isolated by reversed phase liquid chromatography
and the final dimethoxytrityl group on the 5'-end of the probe will be removed by
treatment with 80s acetic acid to yield the purified ANA probe, which is ready for
use in the hybridization assay.
C. Hybridization Procedure
[0027] It is envisioned that the protocol for use of A
NA probes would follow along the lines of the various hybridization assays commonly
utilized and no limitations on the substitution of ANA probes for DNA or RNA probes
in any system is foreseen. Initially, the target or sample nucleic acid would be prepared
by any convenient procedure. The nucleic acid would be denatured to a single-stranded
state by any conventional means. For example. DNA can be denatured by heating it in
an appropriate buffer at 95°C for 5 minutes. Alternatively, denaturation can be effected
by treating DNA with 0.25 N NaOH for 10 minutes. In this case, following denaturation,
it is necessary to add an equivalent amount of acid (e.g. HCl) to neutralize the solution
containing the single-stranded DNA. At this point, it may also be necessary to adjust
the ionic strength of the sample to optimize the binding of the DNA to a support.
It is advisable to cool the denatured DNA on ice to lower the rate of renaturation
of the single-stranded DNA.
[0028] The target nucleic acid can be immobilized onto the surface of a support. Classically,
the support of choice has been a nitrocellulose membrane. If this material is used,
an aliquot of target nucleic acid can either be spotted onto the membrane or slowly
filtered through the membrane contained in a device such as a dot-blot or slot-blot
manifold. Following application of the target nucleic acid to the nitrocellulose,
the membrane is dried and heated in a vacuum oven at approximately 80
* for 0.5 - 2.0 hours to assure secure attachment to the nitrocellulose.
[0029] The support material is not necessarily limited to nitrocellulose. For example, charged
nylon supports such as Gene Screen™ (E. I. du Pont de Nemours and Co.. Inc., Wilmington.
DE) or Biotrans™ (I
CN Radiochemicals. Irvine. CA) can also be used. The protocols developed by the manufacturers
of the membranes should be followed when immobilizing nucleic acids. This applies
whether the nucleic acid is being affixed to the membrane in a dot-blot manifold or
by one of the transfer protocols (e.g.. Southern transfer) commonly used after electrophoretic
separation of the sample nucleic acids.
[0030] In a different protocol, the target nucleic acid can be immobilized by hybridizing
it to a strand of "capture" nucleic acid which is immobilized to a support material.
This capture nucleic acid is complementary to a short sequence of bases in the target
nucleic acid and specifically captures it from a solution that may contain substantial
quantities of other nucleic acid which are of no immediate interest.
[0031] The nucleic acid of interest, which has been firmly immobilized to a support material,
is in a single-stranded or denatured state and is available for hybridization with
an ANA probe containing a complementary sequence of bases. The immobilized target
nucleic acid and the support can next be treated with a buffer solution containing
generic DNA (e.g., sonicated salmon sperm DNA) to eliminate nonspecific binding sites
for the specific ANA probe. Typically, this generic DNA is present in the buffer at
a concentration of 100 µg/mi and 100 µl of this prehybridization buffer is required
for each cm
2 of support material. This prehybridization buffer can also contain 10% sodium dextran
sulfate, 0.1% sodium dodecyl sulfate, 50% formamide, and SSPE, which is a mixture
of sodium chloride, sodium phosphate, and EDTA. In addition, the buffer can also contain
Denhardt's reagent, which is a mixture of ficoll. polyvinyl pyrollidone, and bovine
serum albumin.
[0032] The support to which the nucleic acid is immobilized is prehybridized in this buffer
mixture at an elevated temperature (e.g.. 37° - 65°C) for a period of time ranging
from several hours to overnight in an effort to block sites on the immobilized nucleic
acid to which the ANA probe can be attached nonspecifically. Following prehybridization.
ANA probe is added to the prehybridization buffer to the desired final concentration,
typically 10 - 100 ng/mL. Hybridization is then carried out at the appropriate elevated
temperature (e.g., 37° - 65°C) for an appropriate period of time. usually overnight.
[0033] Following hybridization, the support is washed with a series of buffers to remove
ANA probe which may be nonspecifically attached to the support. Typically, as this
sequence of washes progresses, the concentration of the salt in the buffer is reduced
and the temperature of the wash is increased. Following this series of washes, the
support is then rinsed with the appropriate buffer to remove any reagents from the
wash buffers that may have a detrimental effect on the activity of the antibody-enzyme
conjugate to be added later.
D. production of Antibodies
[0034] Either polyclonal or monoclonal antibodies can be used to detect arabinose in the
ANA probe molecules. Polyclonal antibodies can be produced by any convenient method
used to produce antibodies to modified DNA [e.g.. S. Cohn and M. W. Lieberman. J.
Biol. Chem. Volume 259, 12456-62 (1984)]. Likewise, monoclonal antibodies can be produced
by any of a number of procedures (H. G. Gratzner, Science Volume 218. 474-5 (1982)].
[0035] It is believed that an arabinonucleotide conjugated to a suitable carrier protein
(e.g. BSA) can also serve as the immunogen in antibody production. However, the ANA
probe alone (or hybridized to complementary nucleic acid) would be preferred. Alternatively,
the probe can be conjugated to a carrier protein to serve as the immunogen. Okabayashi
et al. [Cancer Research Volume 37. 619-624 (1977)] reported production of antibodies
to 1-B-D-arabinofuranosyl- cytosine (ara-C) in plasma. These antibodies did not cross-react
with deoxycytidine or cytidine which differ from ara-C only in the 2
1-position. This suggests very strongly that antibodies of suitable specificity for
arabinose in ANA, which will not cross-react with deoxyribose in DNA or ribose in
RNA, can be developed.
E. Preparation of the Antibody-Enzyme ConiuQate
[0036] Conjugation of the antibody to an enzyme can be carried out by known methods. For
example, the use of glutaraldehyde to link amino groups on the enzyme and the antibody
is a common approach [H. Wallin, et al.. Cancer Letters Volume 22, 163-170 (1984)].
Using this protocol, the enzyme (e.g. peroxidase) is allowed to react for 18 hours
at room temperature with glutaraldehyde. After removal of excess glutaraldehyde in
a gel filtration column, the activated enzyme is allowed to react with the antibody
for 24 hours at 4°C. The antibody-enzyme conjugate is then purified by dialysis, ammonium
sulfate precipitation, and gel filtration chromatography. Conjugation procedures using
heterobifunctional crosslinking agents such as those described by J. W. Freytag et
al.. Clin. Chem., Volume 30. 417-420 (1984). or C. C. Leflar et al.. Clin. Chem.,
Volume 30, 1809-1811 (1984) can also be used. It is expected that peroxidase. B-galactosidase,
glucose oxidase, alkaline or acid phosphatase or any other useful enzyme could be
conjugated to the antibody (or a fragment thereof) and subsequently used to detect
arabinose in the ANA probe.
F. Detection of Hybrids
[0037] The hybridized sample would be incubated in a blocking buffer which contains reagents
to reduce nonspecific adsorption of the antibody-enzyme conjugate to the membrane.
Typically, a buffer containing 1% bovine serum albumin is used. Following treatment
with the blocking buffer, the membrane would be incubated with an appropriate antibody-enzyme
conjugate for a period of time such that the antibody has an opportunity to recognize
and bind to each arabinose moiety in the strands of probe hybridized to target nucleic
acid. The antibody-enzyme conjugate would consist of an enzyme covalently linked to
an antibody or fragment thereof which is specific for arabinose in the probe.
[0038] Following binding of the antibody-enzyme conjugate to the probe, the membrane. which
now contains conjugate bound to the probe hybridized to the target nucleic acid. is
washed again with a series of buffers to remove unbound and nonspecifically bound
conjugate from the support. The appropriate enzyme substrate and/or chromogen is incubated
with the product on the support and color development is allowed to proceed for a
prescribed period of time. The extent of hybridization could be quantified by measuring
the rate of color development or the total color developed after a set time period.